Function of Fe(III)-minerals in the enhancement of anammox performance exploiting integrated network and metagenomics analyses

Iron is a recognized physiological requirement for microorganisms but, for anaerobic ammonium oxidation (anammox) bacteria, its role extends well beyond that of a nutritional necessity. In this study, the function of two typical Fe(III)-minerals (ferrihydrite and magnetite) in anammox processes was evaluated in the absence/presence of Fe(II) by integrated network and metagenomics analyses. Results showed that Fe-(III) minerals addition increased the activity of cellular processes and pathways associated with granule formation, enabling the peak values of particle size to increase by 144% and 115%, respectively. Notably, ferrihydrite (5 mM) enhanced nitrogen removal by 4.8% and 4.1%, respectively, in the short-term and long-term absence of Fe(II). Ferrihydrite also promoted the retention of anammox bacteria affiliated with phylum Planctomycetes in the reactor, contributing to an 11% higher abundance with ferrihydrite amendment when compared with the control (without iron additions) in the short-term absence of Fe(II). Network-based analyses revealed that ferrihydrite facilitated the microbial community to form densely clustered and complex topologies to improve resistance to environmental disturbance (i.e., Fe(II) deficiency), and effectively increased the underlying cooperation and facilitation in the community. Metagenomic analysis revealed that there was limited promotion of anammox central metabolism by the extra addition of Fe(III)-minerals in the presence of Fe(II), highlighting the poor utilization of Fe(III)-minerals by anammox bacteria under Fe(II) sufficiency. This study deepens our understanding of the function of Fe(III)-minerals in anammox systems at the community and functional level, and provides a fundamental basis for developing Fe-based anammox enhancement technologies.